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Determination of polynuclear aromatic hydrocarbons in human blood serum by proteolytic digestion — direct immersion SPME
Analytica Chimica Acta 396 (1999) 303–308
Determination of polynuclear aromatic hydrocarbons in human blood serum by proteolytic digestion — direct immersion SPME Ka-Fai Poon, Paul K.S. Lam, Michael H.W. Lam ∗ Centre for Coastal Pollution and Conservation, Department of Biology & Chemistry, City University of Hong Kong, Tat Chee Ave., Kowloon, Hong Kong Received 3 May 1999; received in revised form 7 May 1999; accepted 25 May 1999
Abstract A proteolytic digestion — direct immersion solid phase microextraction (SPME) method has been developed for the determination of polynuclear aromatic hydrocarbons in human blood serum. Non-specific serine protease, Proteinase K, was employed to remove the fouling of the SPME stationary phase by the lipoprotein in blood serum during extraction. Optimization of protease concentration and duration of the proteolytic digestion as well as SPME sampling have been performed. Proteinase K concentration of 190 g protease/ml of serum and digestion time of 60 min at 25◦ C were found to be the optimum conditions for the determination. PAH recoveries of the direct immersion SPME method ranged from 81.1 to 98.5%. The relative repeatability of the method was found to be 5.6% (n = 10). Detection limits for the 16 prioritized PAHs ranged from 2.7 to 30.4 ppb. ©1999 Elsevier Science B.V. All rights reserved. Keywords: SPME; PAH; Human blood serum; Proteolytic digestion
1. Introduction With the rapid development in separation science and instrumentation, most modern analytical instruments nowadays are sensitive enough to detect analytes down to pico- or even fento-gram levels. Due to that, efficiencies of sample extraction and cleanup steps are becoming increasingly significant in restraining detection limits of analytical methods [1]. One of the research objectives of our laboratory is to develop new, more efficient and reliable extraction ∗ Corresponding author. Tel.: +852-2788-7329; fax: +852-2788-7406 E-mail address: [email protected] (M.H.W. Lam)
methods for the determination of organic pollutants in biological tissues and fluids. The growing popularity of utilizing solid sorbents for the selective adsorption/absorption of analytes from solution and vapour phases has led us to examine the applicability of solid phase microextraction (SPME) for the extraction of organic pollutants from human blood serum. Pioneered by Pawliszyn et. al. [2–5], the SPME technique involves the partitioning of organic analytes in the aqueous or gaseous medium onto the stationary phase coated on a thin fused silica fibre. The extracted analytes can be determined by GC via thermal desorption at the GC injector port, or by HPLC via special interface. Advantages of SPME include completely solvent-free extraction, high pre-concentration
0003-2670/99/$ – see front matter ©1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 9 9 ) 0 0 4 4 7 - X
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K.-F. Poon et al. / Analytica Chimica Acta 396 (1999) 303–308
factor, no need of clean-up procedures and simple instrumentation. While very efficient extraction of organic analytes in aqueous media such as natural water and wastewater can be achieved by SPME, the method becomes inefficient in more complicated sample matrices such as oily or greasy water and biological fluids. In these matrices, the SPME stationary phase coating is rapidly fouled by greases and protein molecules. As fouling occurs simultaneously with analyte absorption, changes in the surface area and nature of the SPME stationary phase render the SPME determination unreliable and irreproducible. Currently, most of the SPME procedures developed for applications in body fluid matrices, especially blood, involve headspace sampling [6–10]. In this way, fouling of the stationary phase coating can be avoided. However, headspace SPME is not without its limitations. Firstly, headspace sampling reduces the amount of analytes absorbed by the SPME coating, which in turn increases the method detection limit. This can only be alleviated by increasing the extraction time and the extraction temperature, which may not be desirable in terms of cost efficiency of analysis and stability of some analytes at higher temperature, respectively. Secondly, the extraction efficiency of headspace SPME depends strongly on the KOW values and Henry’s constants KH , of analytes [11]. Recently, we developed a direct immersion SPME method applicable to biological fluid matrix such as human blood serum. The method involves a ‘deproteination’ step using a non-specific serine protease, Proteinase K. In this work, we report such a direct immersion SPME technique for the determination of the 16 prioritized polynuclear aromatic hydrocarbons in human blood serum.
2. Experimental PAH standards were obtained from Aldrich. Acetone (pesticide grade, Aldrich) was used to promote solubility of the PAHs in the serum. In any case, acetone in the serum sample was below 1%. Human blood serum was obtained by centrifugation of whole blood sample. The PAH standards were spiked into the serum sample immediately after centrifugation. No protein precipitation nor denaturation steps were performed prior to PAH addition. Proteinase K (fun-
gal) was obtained from Life Technologies. The SPME device (with 100 m polydimethylsiloxane coating) was obtained from Supelco Inc. A Hewlett Packard HP-5890II GC with a 50 m × 0.2 mm × 0.33 m HP-5 (trace analysis) (5% PHME Siloxane) column and a flame ionization detector was used for the PAH determination. Protease reconstitution was carried out by dissolving the Proteinase K in 50 ml of water containing 20 mM calcium chloride, 10 mM Tris–HCl and 50% glycerol. The pH of the solution was adjusted to 7.5 (with Tris–base) and the solution was stored at −20◦ C before use. The SPME fibre was pre-conditioned at 280◦ C under helium overnight prior to its first extraction. Thermal desorption of the extracted PAHs was effected by inserting the SPME fibre into the GC injector port under splitless mode (5.25 min), kept at 270◦ C, for 5.0 min. The GC column temperature was first held at 40◦ C for 20 min and increased to 200◦ C at a rate of 7◦ C/min. The column temperature was held at 200◦ C for 5 min and then raised to 280◦ C at a rate of 9◦ C/min. After holding at 280◦ C for 14 min, the column temperature was further raised to 290◦ C at a rate of 7◦ C/min. After each thermal desorption, the SPME fibre was re-conditioned by keeping it in the GC injection port for 30 min at 280◦ C. The fibre blank was checked by putting the conditioned SPME fibre in clean deionized water for 15 min followed by thermal desorption and GC analysis before each extraction. Throughout the study, no PAH residues were found to be left on the SPME fibre after each re-conditioning. In a typical SPME determination, 3.8 ml of blood serum sample was spiked with known amount of PAH standards and was mixed with 0.4 ml protease solution. The mixture was then placed in a 10 ml clean vial and was sealed and stirred at 25◦ C. After the protease digestion, a conditioned SPME fibre was immersed into the resultant sample mixture extraction. Extraction time was 60 min during which stirring was maintained. After the extraction, the SPME fibre was rinsed in 0.9% NaCl solution (10 s) followed by deionized water (10 s) before thermal desorption and GC determination. Recovery of the direct immersion SPME determination was determined by comparing the SPME/GC-FID respond of the blood serum sample to that obtained from the control with 3.8 ml saline solution containing similar spike levels of protease and PAH standards.
K.-F. Poon et al. / Analytica Chimica Acta 396 (1999) 303–308
305
Fig. 1. Total SPME/GC-FID response of all the 16 prioritized PAHs in blood serum at various Proteinase K concentrations. Spike levels of PAHs ranged from 0.2 to 3.8 ppm. Duration of protease digestion and SPME sampling were 30 min and 60 min, respectively.
3. Results and discussion Severe fouling can readily be observed by directly immersing a SPME fibre into an undeproteinated blood serum sample. Building up of protein coating (presumably albumins and globulins which constitute approximately 2% w/v of human plasma) on the SPME fibre is visible within 5 min. Besides the reduction in the extraction repeatability and reproducibility, the protein coating formed was not totally removable by rinsing, denaturation and sonification and was still attached to the SPME fibre even after thermal desorption at the GC injector port. This seriously reduced the reusability of the SPME device. Common protein denaturation/precipitation methods, such as heating, treatment with methanol, trifluoroacetic acid or perchloric acid [12] have been tried to remove the lipoproteins in the blood serum sample before the solid phase microextraction. All treatments ended up with less than 50% recoveries for all the PAHs. The low recovery may be attributable to the scavenging of PAHs in the serum by the coagulation and precipitation of the denatured lipoproteins. While chemical denaturation approaches were found unfeasible for direct immersion SPME determination in the serum matrix, an enzymatic proteolytic approach was tried. A non-specific serine protease, Proteinase K, commonly used to inactivate endo-
genous nucleases during RNA/DNA isolation [13], was used to breakdown the lipoproteins in the serum into amino acid fragments before direct immersion SPME determination. Proteinase K was selected for this application as it was able to retain its activity even in the presence of metal ions, chelating agent, sulfhydryl reagents or by trypsin or chymotrypsin inhibitors. It was also stable over wide pH range (from pH 4 to 12.5 with the optimum pH in the range 6.5–9.5) [14]. No build-up of protein coating on the SPME device was observed after the implementation of protease digestion. Fig. 1 shows the relation between extraction efficiency of the direct immersion SPME (in terms of the total area count of all the 16 prioritized PAHs) and the concentration of Proteinase K. An optimum Proteinase K concentration for the protease digestion was found at 190 g protease/ml of serum. The existence of an optimum protease concentration can be interpreted as follow: Below the optimum protease concentration, undigested lipoproteins in the sample can still interfere with the SPME process and reduce the amount of PAHs absorbed. Conversely, at a Proteinase K concentration higher than the optimum, the protease itself may contribute to the fouling interference. Fig. 2 shows the relation between the SPME efficiency and the duration of protease digestion at 25◦ C. The relationship followed a simple exponential association
306
K.-F. Poon et al. / Analytica Chimica Acta 396 (1999) 303–308
Fig. 2. Total SPME/GC-FID response of all the 16 prioritized PAHs in blood serum at various durations of protease digestion. Spike levels of PAHs ranged from 0.2 to 3.8 ppm. Concentration of Proteinase K was 190 g/ml serum. Duration of SPME sampling was 60 min. Table 1 Recoveries and detection limits of the Proteinase K — direct immersion SPME method for the 16 prioritized PAHs in human blood serum PAHa
Detection limit (ppb)d
Recovery with Proteinase K(%)
Recovery without Proteinase K(%)
Naphthaleneb Acenaphthylenec Acenaphtheneb Fluorenec Phenanthreneb Anthraceneb Fluoranthenec Pyreneb Benzo(a)anthraceneb Chryseneb Benzo(b)fluoranthenec Benzo(k)fluorantheneb Benzo(a)pyreneb Indeno(1,2,3-cd)pyreneb Dibenzo(ah)anthracenec Benzo(ghi)perylenec
6.6 2.7 3.0 6.6 3.4 3.7 3.6 2.1 16.9 7.6 9.3 10.8 3.7 27.8 30.4 14.1
82.1 94.7 82.3 87.6 81.1 89.9 85.4 90.2 93.2 80.1 86.6 84.5 98.5 83.5 83.4 89.6
38.5 32.4 31.0 53.1 63.9 45.3 60.3 52.0 58.3 51.0 46.9 53.2 61.9 48.5 41.6 24.6
Volume of blood serum sample = 3.8 ml, concentration of Proteinase K = 190 g/ml serum, duration of protease digestion = 30 min, SPME sampling time = 60 min. b Spike level = 88 ppb. c Spike level = 177 ppb. d n = 5; P > 0.05. a
with a rate constant of 0.039 s−1 . Maximum SPME efficiency was achieved after ca. 60 min of digestion at 25◦ C. This means that the proteolytic digestion of the lipoprotein in the serum sample is completed within 60 min at room temperature. There was no evidence that prolonged protease digestion lowered the extraction efficiency. Higher digestion temperature was not
attempted in view of the possibility of denaturing of the blood lipoprotein. Improvement in the PAH recoveries by the proteolytic digestion is demonstrated in Table 1. Significant improvement in PAH recoveries from 24.6–61.9% (without proteolytic digestion) to 81.1–98.5% by the Proteinase K digestion were observed. The implications of such improvement are
K.-F. Poon et al. / Analytica Chimica Acta 396 (1999) 303–308
307
308
K.-F. Poon et al. / Analytica Chimica Acta 396 (1999) 303–308
twofold: (a) it confirms the importance of lipoproteins in blood serum as interference to the SPME process, and (b) it demonstrates the effectiveness of Proteinase K as a proteolytic digestion agent. The PAH recoveries of the Proteinase K — direct immersion SPME are comparable to those achievable by conventional solvent extraction and cloud-point extraction approaches [15–17]. At PAH spike levels ranging from 0.2 to 3.8 ppm and Proteinase K concentration of 190 g/ml of serum, the relative repeatability 1 of the Proteinase K — direct immersion SPME determination was found to be 5.6% (the mean GC-FID response, was 1.45 × 105 counts with σ = 1.14 × 104 counts in 10 consecutive determinations). Such repeatability is also comparable to most conventional PAH determination methods in blood serum matrix. Absorption – time profiles of the 16 PAHs are shown in Fig. 3(A-D) . For all PAHs, the amount extracted by the SPME fibre surpassed 90% of the corresponding equilibrium values within 60 min. Table 1 also tabulates the detection limits of the Proteinase K — direct immersion SPME method for the 16 prioritized PAHs in human blood serum. The detection limits ranged from 2.7 to 30.4 ppb. In general, smaller molecular mass PAHs shows lower detection limits.
4. Conclusions The capability of enzymatic proteolytic digestion in removing interference from blood lipoproteins during direct immersion SPME determination of PAHs in blood serum has been demonstrated. Simple protein digestion pretreatment of the blood serum sample by a non-specific serine protease, Protease K, at room temperature is enough to remove most of the matrix interferences. Repeatabilities, analyte recoveries and detection limits achieved for the 16 prioritized PAHs are comparable to conventional solvent extraction methods. In addition, the present SPME approach possesses advantages, such as very simple
2.26σ √ × 100 x¯ n where x¯ is the mean result, σ the standard deviation of the results and n the number of independent extractions. 1
Relative repeatability =
extraction procedures and no need of potentially health hazardous and environmental unfriendly organic solvents. Our work demonstrated that the Proteinase K — direct immersion SPME method is a feasible alternative to conventional solvent extraction methods as well as headspace SPME method for the determination of hydrophobic organic pollutants in blood serum matrix.
References [1] L.H. Keith, Environmental Sampling and Analysis: A Practical Guide, Lewis Publishers Inc., Chelsea, MI, 1992. [2] C.L. Arthur, J. Pawliszyn, Anal. Chem. 62 (1990) 2145. [3] C.L. Arthur, L.M. Killam, K.D. Buchholz, J. Pawliszyn, Anal. Chem. 64 (1992) 1960. [4] D.S. Louch, S. Motlagh, J. Pawliszyn, Anal. Chem. 64 (1992) 1187–1199. [5] Z. Zhang, M. Yang, J. Pawliszyn, Anal. Chem. 66 (1994) 844A. [6] A. Namera, M. Yashiki, N. Nagasawa, Y. Iwasaki, T. Kojima, Forensic Sci. Int. 88 (1997) 125. [7] T. Watanabe, A. Namera, M. Yashiki, Y. Iwasaki, T. Kojima, J. Chromatogr. B 709 (1998) 225. [8] T. Watanabe, A. Namera, M. Yashiki, J. Chromatogr. B 709 (1998) 225. [9] F.Y. Guan, K. Watanabe, A. Ishii, J. Chromatogr. B 714 (1998) 205. [10] K. Takekawa, M. Oya, A. Kido, Chromatographia 47 (1998) 209. [11] Z. Zhang, Pawliszyn. J., Anal. Chem. 65 (1993) 1843. [12] R.V. Smith, J.T. Stewart, Textbook of Biopharmaceutic Analysis: A Description of Methods for the Determination of Drugs in Biological Fluids, Lea & Febiger, London, 1981. [13] S.S. Nikaido, C.R. Locke, D.P. Weeks, Plant Mol. Biol. 26 (1994) 275. [14] W. Ebeling, N. Hennrich, M. Klockow, H. Metz, H.D. Orth, H. Lang, Eur. J. Biochem. 47 (1974) 91. [15] W.H. Griest, J.E. Caton, in: A. Bisørseth (Ed.), Handbook of Polycyclic Aromatic Hydrocarbons, Marcel Dekker, New York, 1983. [16] F.J. van Schooten, E.J.C. Moonen, L. van der Wal, P. Levels, J.C.S. Kleinjans, Arch. Environ. Contamin. Toxicol. 33 (1997) 317. [17] S.R. Sirimanne, J.R. Barr, D.G. Patterson Jr., Anal. Chem. 68 (1996) 1556.
Determination of polynuclear aromatic hydrocarbons in human blood serum by proteolytic digestion — direct immersion SPME Ka-Fai Poon, Paul K.S. Lam, Michael H.W. Lam ∗ Centre for Coastal Pollution and Conservation, Department of Biology & Chemistry, City University of Hong Kong, Tat Chee Ave., Kowloon, Hong Kong Received 3 May 1999; received in revised form 7 May 1999; accepted 25 May 1999
Abstract A proteolytic digestion — direct immersion solid phase microextraction (SPME) method has been developed for the determination of polynuclear aromatic hydrocarbons in human blood serum. Non-specific serine protease, Proteinase K, was employed to remove the fouling of the SPME stationary phase by the lipoprotein in blood serum during extraction. Optimization of protease concentration and duration of the proteolytic digestion as well as SPME sampling have been performed. Proteinase K concentration of 190 g protease/ml of serum and digestion time of 60 min at 25◦ C were found to be the optimum conditions for the determination. PAH recoveries of the direct immersion SPME method ranged from 81.1 to 98.5%. The relative repeatability of the method was found to be 5.6% (n = 10). Detection limits for the 16 prioritized PAHs ranged from 2.7 to 30.4 ppb. ©1999 Elsevier Science B.V. All rights reserved. Keywords: SPME; PAH; Human blood serum; Proteolytic digestion
1. Introduction With the rapid development in separation science and instrumentation, most modern analytical instruments nowadays are sensitive enough to detect analytes down to pico- or even fento-gram levels. Due to that, efficiencies of sample extraction and cleanup steps are becoming increasingly significant in restraining detection limits of analytical methods [1]. One of the research objectives of our laboratory is to develop new, more efficient and reliable extraction ∗ Corresponding author. Tel.: +852-2788-7329; fax: +852-2788-7406 E-mail address: [email protected] (M.H.W. Lam)
methods for the determination of organic pollutants in biological tissues and fluids. The growing popularity of utilizing solid sorbents for the selective adsorption/absorption of analytes from solution and vapour phases has led us to examine the applicability of solid phase microextraction (SPME) for the extraction of organic pollutants from human blood serum. Pioneered by Pawliszyn et. al. [2–5], the SPME technique involves the partitioning of organic analytes in the aqueous or gaseous medium onto the stationary phase coated on a thin fused silica fibre. The extracted analytes can be determined by GC via thermal desorption at the GC injector port, or by HPLC via special interface. Advantages of SPME include completely solvent-free extraction, high pre-concentration
0003-2670/99/$ – see front matter ©1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 9 9 ) 0 0 4 4 7 - X
304
K.-F. Poon et al. / Analytica Chimica Acta 396 (1999) 303–308
factor, no need of clean-up procedures and simple instrumentation. While very efficient extraction of organic analytes in aqueous media such as natural water and wastewater can be achieved by SPME, the method becomes inefficient in more complicated sample matrices such as oily or greasy water and biological fluids. In these matrices, the SPME stationary phase coating is rapidly fouled by greases and protein molecules. As fouling occurs simultaneously with analyte absorption, changes in the surface area and nature of the SPME stationary phase render the SPME determination unreliable and irreproducible. Currently, most of the SPME procedures developed for applications in body fluid matrices, especially blood, involve headspace sampling [6–10]. In this way, fouling of the stationary phase coating can be avoided. However, headspace SPME is not without its limitations. Firstly, headspace sampling reduces the amount of analytes absorbed by the SPME coating, which in turn increases the method detection limit. This can only be alleviated by increasing the extraction time and the extraction temperature, which may not be desirable in terms of cost efficiency of analysis and stability of some analytes at higher temperature, respectively. Secondly, the extraction efficiency of headspace SPME depends strongly on the KOW values and Henry’s constants KH , of analytes [11]. Recently, we developed a direct immersion SPME method applicable to biological fluid matrix such as human blood serum. The method involves a ‘deproteination’ step using a non-specific serine protease, Proteinase K. In this work, we report such a direct immersion SPME technique for the determination of the 16 prioritized polynuclear aromatic hydrocarbons in human blood serum.
2. Experimental PAH standards were obtained from Aldrich. Acetone (pesticide grade, Aldrich) was used to promote solubility of the PAHs in the serum. In any case, acetone in the serum sample was below 1%. Human blood serum was obtained by centrifugation of whole blood sample. The PAH standards were spiked into the serum sample immediately after centrifugation. No protein precipitation nor denaturation steps were performed prior to PAH addition. Proteinase K (fun-
gal) was obtained from Life Technologies. The SPME device (with 100 m polydimethylsiloxane coating) was obtained from Supelco Inc. A Hewlett Packard HP-5890II GC with a 50 m × 0.2 mm × 0.33 m HP-5 (trace analysis) (5% PHME Siloxane) column and a flame ionization detector was used for the PAH determination. Protease reconstitution was carried out by dissolving the Proteinase K in 50 ml of water containing 20 mM calcium chloride, 10 mM Tris–HCl and 50% glycerol. The pH of the solution was adjusted to 7.5 (with Tris–base) and the solution was stored at −20◦ C before use. The SPME fibre was pre-conditioned at 280◦ C under helium overnight prior to its first extraction. Thermal desorption of the extracted PAHs was effected by inserting the SPME fibre into the GC injector port under splitless mode (5.25 min), kept at 270◦ C, for 5.0 min. The GC column temperature was first held at 40◦ C for 20 min and increased to 200◦ C at a rate of 7◦ C/min. The column temperature was held at 200◦ C for 5 min and then raised to 280◦ C at a rate of 9◦ C/min. After holding at 280◦ C for 14 min, the column temperature was further raised to 290◦ C at a rate of 7◦ C/min. After each thermal desorption, the SPME fibre was re-conditioned by keeping it in the GC injection port for 30 min at 280◦ C. The fibre blank was checked by putting the conditioned SPME fibre in clean deionized water for 15 min followed by thermal desorption and GC analysis before each extraction. Throughout the study, no PAH residues were found to be left on the SPME fibre after each re-conditioning. In a typical SPME determination, 3.8 ml of blood serum sample was spiked with known amount of PAH standards and was mixed with 0.4 ml protease solution. The mixture was then placed in a 10 ml clean vial and was sealed and stirred at 25◦ C. After the protease digestion, a conditioned SPME fibre was immersed into the resultant sample mixture extraction. Extraction time was 60 min during which stirring was maintained. After the extraction, the SPME fibre was rinsed in 0.9% NaCl solution (10 s) followed by deionized water (10 s) before thermal desorption and GC determination. Recovery of the direct immersion SPME determination was determined by comparing the SPME/GC-FID respond of the blood serum sample to that obtained from the control with 3.8 ml saline solution containing similar spike levels of protease and PAH standards.
K.-F. Poon et al. / Analytica Chimica Acta 396 (1999) 303–308
305
Fig. 1. Total SPME/GC-FID response of all the 16 prioritized PAHs in blood serum at various Proteinase K concentrations. Spike levels of PAHs ranged from 0.2 to 3.8 ppm. Duration of protease digestion and SPME sampling were 30 min and 60 min, respectively.
3. Results and discussion Severe fouling can readily be observed by directly immersing a SPME fibre into an undeproteinated blood serum sample. Building up of protein coating (presumably albumins and globulins which constitute approximately 2% w/v of human plasma) on the SPME fibre is visible within 5 min. Besides the reduction in the extraction repeatability and reproducibility, the protein coating formed was not totally removable by rinsing, denaturation and sonification and was still attached to the SPME fibre even after thermal desorption at the GC injector port. This seriously reduced the reusability of the SPME device. Common protein denaturation/precipitation methods, such as heating, treatment with methanol, trifluoroacetic acid or perchloric acid [12] have been tried to remove the lipoproteins in the blood serum sample before the solid phase microextraction. All treatments ended up with less than 50% recoveries for all the PAHs. The low recovery may be attributable to the scavenging of PAHs in the serum by the coagulation and precipitation of the denatured lipoproteins. While chemical denaturation approaches were found unfeasible for direct immersion SPME determination in the serum matrix, an enzymatic proteolytic approach was tried. A non-specific serine protease, Proteinase K, commonly used to inactivate endo-
genous nucleases during RNA/DNA isolation [13], was used to breakdown the lipoproteins in the serum into amino acid fragments before direct immersion SPME determination. Proteinase K was selected for this application as it was able to retain its activity even in the presence of metal ions, chelating agent, sulfhydryl reagents or by trypsin or chymotrypsin inhibitors. It was also stable over wide pH range (from pH 4 to 12.5 with the optimum pH in the range 6.5–9.5) [14]. No build-up of protein coating on the SPME device was observed after the implementation of protease digestion. Fig. 1 shows the relation between extraction efficiency of the direct immersion SPME (in terms of the total area count of all the 16 prioritized PAHs) and the concentration of Proteinase K. An optimum Proteinase K concentration for the protease digestion was found at 190 g protease/ml of serum. The existence of an optimum protease concentration can be interpreted as follow: Below the optimum protease concentration, undigested lipoproteins in the sample can still interfere with the SPME process and reduce the amount of PAHs absorbed. Conversely, at a Proteinase K concentration higher than the optimum, the protease itself may contribute to the fouling interference. Fig. 2 shows the relation between the SPME efficiency and the duration of protease digestion at 25◦ C. The relationship followed a simple exponential association
306
K.-F. Poon et al. / Analytica Chimica Acta 396 (1999) 303–308
Fig. 2. Total SPME/GC-FID response of all the 16 prioritized PAHs in blood serum at various durations of protease digestion. Spike levels of PAHs ranged from 0.2 to 3.8 ppm. Concentration of Proteinase K was 190 g/ml serum. Duration of SPME sampling was 60 min. Table 1 Recoveries and detection limits of the Proteinase K — direct immersion SPME method for the 16 prioritized PAHs in human blood serum PAHa
Detection limit (ppb)d
Recovery with Proteinase K(%)
Recovery without Proteinase K(%)
Naphthaleneb Acenaphthylenec Acenaphtheneb Fluorenec Phenanthreneb Anthraceneb Fluoranthenec Pyreneb Benzo(a)anthraceneb Chryseneb Benzo(b)fluoranthenec Benzo(k)fluorantheneb Benzo(a)pyreneb Indeno(1,2,3-cd)pyreneb Dibenzo(ah)anthracenec Benzo(ghi)perylenec
6.6 2.7 3.0 6.6 3.4 3.7 3.6 2.1 16.9 7.6 9.3 10.8 3.7 27.8 30.4 14.1
82.1 94.7 82.3 87.6 81.1 89.9 85.4 90.2 93.2 80.1 86.6 84.5 98.5 83.5 83.4 89.6
38.5 32.4 31.0 53.1 63.9 45.3 60.3 52.0 58.3 51.0 46.9 53.2 61.9 48.5 41.6 24.6
Volume of blood serum sample = 3.8 ml, concentration of Proteinase K = 190 g/ml serum, duration of protease digestion = 30 min, SPME sampling time = 60 min. b Spike level = 88 ppb. c Spike level = 177 ppb. d n = 5; P > 0.05. a
with a rate constant of 0.039 s−1 . Maximum SPME efficiency was achieved after ca. 60 min of digestion at 25◦ C. This means that the proteolytic digestion of the lipoprotein in the serum sample is completed within 60 min at room temperature. There was no evidence that prolonged protease digestion lowered the extraction efficiency. Higher digestion temperature was not
attempted in view of the possibility of denaturing of the blood lipoprotein. Improvement in the PAH recoveries by the proteolytic digestion is demonstrated in Table 1. Significant improvement in PAH recoveries from 24.6–61.9% (without proteolytic digestion) to 81.1–98.5% by the Proteinase K digestion were observed. The implications of such improvement are
K.-F. Poon et al. / Analytica Chimica Acta 396 (1999) 303–308
307
308
K.-F. Poon et al. / Analytica Chimica Acta 396 (1999) 303–308
twofold: (a) it confirms the importance of lipoproteins in blood serum as interference to the SPME process, and (b) it demonstrates the effectiveness of Proteinase K as a proteolytic digestion agent. The PAH recoveries of the Proteinase K — direct immersion SPME are comparable to those achievable by conventional solvent extraction and cloud-point extraction approaches [15–17]. At PAH spike levels ranging from 0.2 to 3.8 ppm and Proteinase K concentration of 190 g/ml of serum, the relative repeatability 1 of the Proteinase K — direct immersion SPME determination was found to be 5.6% (the mean GC-FID response, was 1.45 × 105 counts with σ = 1.14 × 104 counts in 10 consecutive determinations). Such repeatability is also comparable to most conventional PAH determination methods in blood serum matrix. Absorption – time profiles of the 16 PAHs are shown in Fig. 3(A-D) . For all PAHs, the amount extracted by the SPME fibre surpassed 90% of the corresponding equilibrium values within 60 min. Table 1 also tabulates the detection limits of the Proteinase K — direct immersion SPME method for the 16 prioritized PAHs in human blood serum. The detection limits ranged from 2.7 to 30.4 ppb. In general, smaller molecular mass PAHs shows lower detection limits.
4. Conclusions The capability of enzymatic proteolytic digestion in removing interference from blood lipoproteins during direct immersion SPME determination of PAHs in blood serum has been demonstrated. Simple protein digestion pretreatment of the blood serum sample by a non-specific serine protease, Protease K, at room temperature is enough to remove most of the matrix interferences. Repeatabilities, analyte recoveries and detection limits achieved for the 16 prioritized PAHs are comparable to conventional solvent extraction methods. In addition, the present SPME approach possesses advantages, such as very simple
2.26σ √ × 100 x¯ n where x¯ is the mean result, σ the standard deviation of the results and n the number of independent extractions. 1
Relative repeatability =
extraction procedures and no need of potentially health hazardous and environmental unfriendly organic solvents. Our work demonstrated that the Proteinase K — direct immersion SPME method is a feasible alternative to conventional solvent extraction methods as well as headspace SPME method for the determination of hydrophobic organic pollutants in blood serum matrix.
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